DNase I and chitosan enhance efficacy of ceftazidime to eradicate Burkholderia pseudomallei biofilm cells

Biofilm-associated Burkholderia pseudomallei infection contributes to antibiotic resistance and relapse of melioidosis. Burkholderia pseudomallei biofilm matrix contains extracellular DNA (eDNA) that is crucial for biofilm establishment. However, the contribution of eDNA to antibiotic resistance by B. pseudomallei remains unclear. In this study, we first demonstrated in vitro that DNase I with the administration of ceftazidime (CAZ) at 24 h considerably inhibited the 2-day biofilm formation and reduced the number of viable biofilm cells of clinical B. pseudomallei isolates compared to biofilm treated with CAZ alone. A 3–4 log reduction in numbers of viable cells embedded in the 2-day biofilm was observed when CAZ was combined with DNase I. Confocal laser-scanning microscope visualization emphasized the competence of DNase I followed by CAZ supplementation to significantly limit B. pseudomallei biofilm development and to eradicate viable embedded B. pseudomallei biofilm cells. Furthermore, DNase I supplemented with chitosan (CS) linked with CAZ (CS/CAZ) significantly eradicated shedding planktonic and biofilm cells. These findings indicated that DNase I effectively degraded eDNA leading to biofilm inhibition and dispersion, subsequently allowing CAZ and CS/CAZ to eradicate both shedding planktonic and embedded biofilm cells. These findings provide efficient strategies to interrupt biofilm formation and improve antibiotic susceptibility of biofilm-associated infections.


DNase I inhibits biofilm formation and enables CAZ to kill B. pseudomallei biofilm cells.
Our previous report showed that DNase I interrupts B. pseudomallei biofilm adhesion and biofilm development 20 . We therefore hypothesized that the degradation of eDNA caused by DNase I interferes with the initial bacterial attachment step and interrupts biofilm formation. This, in turn, should increase CAZ susceptibility of B. pseudomallei cells. The first set of analyses examined the impact of the continuous presence of 0.01, 0.1 or 1 U/mL DNase I followed by adding 512 µg/mL CAZ at 24 h. DNase I alone interfered the biofilm formation as previously demonstrated 20 . The combination of DNase I with CAZ likewise significantly reduced biofilm formation compared to that of untreated control (p < 0.001 in each case) (Fig. 1). The same experiment remarkably improved efficiency of CAZ in eradication of viable B. pseudomallei biofilm cells compared to CAZ alone (p < 0.05 and p < 0.001) (Fig. 2). The most interesting aspect of these data is that only 0.01 U/mL DNase I was required to enable CAZ to bring about a 3-4 log reduction in numbers of B. pseudomallei biofilm cells compared to untreated controls. However, the bacterial susceptibility was not correlated to DNase I concentrations either alone or with CAZ. The DNase I-treated biofilm displayed the inefficiently stained with crystal violet while viable B. pseudomallei were detected. This finding may indicate the distinct biofilm biomass component after treated with DNase I that lack negatively charged biofilm components such as polysaccharides, proteins, or nucleic acids. While B. pseudomallei cells adhered to pegs indicating microcolonies of viable initial biofilms.

DNase I degradation of eDNA resulted in Burkholderia pseudomallei biofilm inhibition and facilitated CAZ killing of embedded biofilm cells. CLSM images of biofilms grown for 48 h in the
presence of 0.01 U/mL DNase I with or without the addition of 512 µg/mL CAZ at 24 h are shown in Fig. 3. A change in morphology of biofilm cells from rod-shaped to filaments and clumps was observed in all three B. pseudomallei strains after treatment with CAZ alone. DNase I-treated biofilms revealed looser biofilm structures and the faint biofilm cells when treated with the combination of CAZ and DNase I.

Live/dead visualization and live/dead ratio of B. pseudomallei biofilm cells treated with DNase I combined with CAZ.
To confirm the competence of DNase I to facilitate CAZ killing activity against embedded B. pseudomallei biofilm cells, live/dead staining was evaluated under CLSM. The results confirmed biofilm erosion. In conjunction with this, the live/dead ratio dropped considerably in all three B. pseudomallei strains compared to that of untreated controls (p < 0.05 in each case) (Fig. 4). Furthermore, DNase I could greatly enhance CAZ killing of B. pseudomallei L1 and H777 biofilm cells compared to CAZ alone (p < 0.001). These data emphasized that DNase I promoted CAZ efficiency leading to CAZ susceptibility of B. pseudomallei biofilm cells. www.nature.com/scientificreports/ There was a remarkable reduction in the biomass of all tested B. pseudomallei biofilms compared to that of untreated controls (p < 0.001) (72-79% reduction), and either CAZ or DNase I alone (p < 0.05 in each case) (Fig. 5). The eDNA in all tested B. pseudomallei biofilms treated with DNase I combined with CAZ was much lower than in the untreated controls (p < 0.001) (52-69% reduction) and less than that treated with CAZ alone in B. pseudomallei L1 and H777 (p < 0.05 and 0.001, respectively). This evidence indicated the effectiveness of DNase I to degrade eDNA leading to biofilm deterioration. DNase I combined with CS/CAZ effectively boosted CAZ killing ability against B. pseudomallei shedding planktonic and biofilm Cells. We next combined DNase I with CS/CAZ, the agent previously reported to improve bactericidal competence against B. pseudomallei biofilms 38 , to test their combined effect against shedding planktonic and embedded biofilm cells. DNase I (0.01 U/mL) and CS/CAZ were combined at various concentrations: 2.5 mg/mL CS/128 µg/mL CAZ, 5 mg/mL CS/256 µg/mL CAZ and 10 mg/mL CS/ 512 µg/mL CAZ. The results revealed that DNase I combined with 10 mg/mL CS/512 µg/mL CAZ completely killed all shed planktonic and biofilm cells (Fig. 6). DNase I combined with CS/CAZ at 2.5 mg/mL CS/128 µg/ mL CAZ and 5 mg/mL CS/256 µg/mL CAZ significantly reduced numbers of both shedding planktonic and biofilm cells compared to untreated controls and CS/CAZ alone (p < 0.05 and 0.001). The most striking result to emerge from the data is that the combination of DNase I and CS/CAZ could kill both planktonic and embedded biofilm cells of B. pseudomallei.
DNase I dispersed the 24-h established biofilm but failed to improve CAZ competence to kill B. pseudomallei biofilm cells. We next investigated the ability of DNase I to disperse and facilitate CAZ killing of the 24-h established B. pseudomallei biofilm. DNase I (0.01, 0.1 or 1 U/mL) with or without 512 µg/ mL CAZ was added to the pre-formed biofilm. Biofilm biomass declined significantly using 0.01 and 0.1 U/mL DNase I combined with CAZ compared to the effect of CAZ alone (p < 0.001) (Fig. 7). However, there was no evidence that DNase I could assist CAZ in the killing of embedded biofilm cells (Fig. 8).

Discussion
Prior studies have noted that Burkholderia pseudomallei biofilm reduces antibiotic susceptibility by limiting antibiotic penetration 23,24 and is correlated with persistent infections 18 . The eDNA, a key constituent of the B. pseudomallei biofilm matrix, is liberated from living biofilm cells 20 . However, the extent to which antibiotic tolerance of B. pseudomallei biofilm is mediated by the presence of eDNA remains to be elucidated. Additionally, bacteria within biofilms are generally more resistant to antibiotics and support the reestablishment of the biofilm construction 39 . Therefore, inhibition of B. pseudomallei biofilm formation, biofilm dispersion and eradication of biofilm cells are all crucial to minimize antibiotic resistance, prevent recurrence and lower mortality rates of lifethreatening melioidosis. Recent research on biofilm resistance has trailed many different compounds to destroy the biofilm matrix and release the planktonic cells to restore the susceptibility to conventional antibiotics 40 . In this study, we extended our previous finding that DNase I degrades eDNA, thus inhibiting and dispersing B. pseudomallei biofilm 20 . We speculated that biofilm inhibition and dispersion will facilitate and enhance CAZ killing of biofilm cells. Our current results further demonstrate the ability of 0.01, 0.1 and 1 U/mL DNase I to degrade eDNA in biofilm matrix, suppress biofilm formation and, crucially, to increase CAZ susceptibility of all three clinical B. pseudomallei isolates. The presence of DNase I improved the susceptibility of B. pseudomallei biofilm cells to 512 µg/mL CAZ. This is a much lower concentration than the previously determined MBEC value for CAZ alone at 2,048 µg/mL 25 . Bactericidal activity was enhanced when 0.01 U/mL DNase I was combined with 512 µg/mL CAZ. Furthermore, the potential antibacterial and antibiofilm properties of CS/CAZ concurred with our initial finding 38 in which CS/CAZ at 2.5 mg/mL CS/128 µg/mL CAZ and 5 mg/mL CS/256 µg/mL CAZ with 0.01 U/mL DNase I significantly improved the efficiency of CAZ to eradicate both shed planktonic and biofilm cells of B. pseudomallei. DNase I could inhibit formation and disrupt the biofilm matrix, allowing the antimicrobial substance to target the detached cells. These findings suggest that DNase I in combination with antimicrobial agents may be a better alternative approach against biofilm-associated pathogens to disperse biofilm and enhance bactericidal efficiency. Extracellular DNA is a key target for dispersing biofilm and improving the vulnerability of biofilm cells to antibiotics 41 . Our results broadly support the work of other studies in this area linking Dnase I with biofilm interruption and increased susceptibility to antimicrobial agents. Li and colleagues revealed that the enzymatic activity of DNase I and dextranase could efficiently reduce biofilm adhesion and improve susceptibility of Enterococcus faecalis biofilms to 2% chlorhexidine 35 . Cavaliere and colleagues found that the presence of the cation chelator ethylenediaminetetra-acetic acid (EDTA) and DNase I destabilized nontypeable Hemophilus influenzae  www.nature.com/scientificreports/ biofilms and enhanced susceptibility to ampicillin and ciprofloxacin 34 . Challenges for the clinical translation of biofilm-dispersing enzymes to avoid detrimental effects in vivo were explored as a novel therapeutic approach for biofilm-associated infections 42 .
The CLSM images of biofilm wiped out consistent with the drop of eDNA and live/dead ratio emphasized the potential of DNase I to degrade eDNA and facilitate CAZ bactericidal competency (Figs. 3, 4). It seems possible that these results are due to the cleavage of eDNA leads to biofilm alteration that increased antibiotic penetration and enhance the efficacy of antibiotic resulted in decrease biofilm biomass and biofilm-associated cell numbers 43 . Use of CAZ alone induced the filamentation of B. pseudomallei cells. The reversible filamentation induced by either sublethal concentrations of CAZ or prolonged antibiotic exposure can possibly affect antibiotic resistance 44 . Moreover, the filamentous appearance of clinical B. pseudomallei CAZ-resistant variants associated with treatment failure during prolonged CAZ therapy of natural infection have been demonstrated 17 . This phenomenon is caused by inhibition of cell division due to inactivation of penicillin-binding protein (PBP)-3 leading to growth into long filaments. Various CAZ concentrations inhibit PBP-3 causing filament formation in Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa and Acinetobacter baumannii, suggesting additional risks during empirical treatment of severe infections 45 .
Recent research on biofilm resistance has focused on different compounds that can destroy the biofilm matrix and release planktonic cells to be attacked by conventional antibiotics 40 . Therefore, the combination of DNase I with antimicrobial agents to eradicate not only biofilm biomass but also biofilm cells and shed planktonic cells would improve the antibiotic susceptibility of biofilm-associated B. pseudomallei infections. We previously showed that DNase I, used in conjunction with an antibacterial and antibiofilm agent, CS/CAZ 38 , significantly improved eradication of biofilm cells and of shed planktonic cells relative to CS/CAZ alone. This observation highlights a potential novel strategy to overcome the inherent resistance of B. pseudomallei biofilms to antibiotics. Our results are consistent with the report of the efficacy of CS gel loaded with solid lipid nanoparticles of silver sulfadiazine supplemented with DNase I against P. aeruginosa biofilm in biofilm-associated wound infection 36 . Clinical studies have demonstrated the ability of recombinant human deoxyribonuclease (rhDNase) to cleave eDNA as a mucolytic agent that can improve mucociliary clearance, increase lung function and reduced the incidence of respiratory-tract infections in cystic fibrosis (CF) 46,47 . Our findings may translate to the use of DNase I combined with CAZ for better treatment of biofilm-associated B. pseudomallei infections.
In general, mature biofilm resists antimicrobial agents by limiting diffusion of these agents into the matrix and by containing persister cells which can survive in the presence of antibiotics 40 . The use of DNase together with CAZ against established biofilm can decrease biofilm formation but not effectively kill the established biofilm cells (Figs. 6, 7). This result may be explained by the fact that effective DNase treatment depends on the age of the biofilm. Young biofilms are simply dispersed but this is not the case for biofilm that has aged beyond a certain point 41 . This suggests that an established biofilm matrix may comprise of additional extracellular polymeric substances including polysaccharides, proteins and lipids, that provide stability 48 and against which DNase I is less effective. DNase I treatment interfered with Listeria monocytogenes biofilm attachment but incompletely  www.nature.com/scientificreports/ dispersed the established biofilm. However, the addition of proteinase K completely dispersed the biofilm. These data suggest that L. monocytogenes biofilm is composed of DNA and proteins 32 . Notably, the combination of trypsin and DNase I effectively function as an anti-biofilm agent against dual-species biofilms of Staphylococcus aureus and Pseudomonas aeruginosa and reduce the MBEC of antibiotics 49 . In addition, combined DNase and proteinase interfere with the composition and structural integrity of multispecies oral biofilms 50 . This may imply that a mixture of enzymes targeting components of the biofilm matrix may be utilized to disperse established biofilms: subsequent supplementation with antibiotics could then kill shedding planktonic cells. However, our preliminary results indicated that there was no significant difference in B. pseudomallei H777 biofilm inhibition or dispersion between untreated controls and treatments using proteinase K. Further work may be required to verify the components of B. pseudomallei biofilm. Overall, DNase I combined with antimicrobial agents could have a great impact on future clinical treatments to prevent biofilm formation, especially for B. pseudomallei. Despite these promising results, questions remain. A note of caution is due here since the high concentration of CAZ may cause difficulties for clinical management. Further studies are required to optimize clinical achievable CAZ concentration and investigate the synergistic activity of DNase I and antimicrobial agents against mature B. pseudomallei biofilm. Thus, the potency of synergistic combinations of DNase I with antimicrobial CS and the drug of choice to treat melioidosis, CAZ, has potential for melioidosis management.

Conclusions
The present study was designed to determine the contribution of eDNA to antibiotic resistance by B. pseudomallei using DNase I. The most obvious finding to emerge from this study is that DNase I degraded eDNA leading to biofilm inhibition and enhanced CAZ efficacy resulted in a 3-4 log reduction in viable B. pseudomallei biofilm cell numbers. The combination of B. pseudomallei shedding planktonic and biofilm cells DNase I with CS/CAZ completely eradicated B. pseudomallei shedding planktonic and biofilm cells. The findings of this study provide a potential therapeutic approach to improve effectiveness treatment against B. pseudomallei biofilm associated infections.  Chitosan-linked ceftazidime (CS/CAZ) preparation. Chitosan from shrimp shells with ≥ 75% deacetylation (Product number C3646, Sigma-Aldrich, Saint Louis, Missouri, USA) (CS) linked to CAZ (CS/CAZ) was prepared as previously described 38 . In brief, 20 mg/mL of CS stock solution was dissolved in 1% v/v acetic acid at 160 rpm overnight at room temperature. The stock solution was adjusted to pH 5.6 before being autoclaved at 121 °C for 20 min and stored at 4 °C until used. The sterile 5000 µg/mL CAZ stock was added dropwise into 20 mg/mL of CS with continuous magnetic stirring at 160 rpm for 24 h to obtain the stock CS/CAZ of 20 mg/mL of CS/1,024 µg/mL CAZ. The solution was used immediately or stored at 4 °C and used within 7 days. On the day of the experiment, the CS/CAZ stock solution was twofold serially diluted to the designated concentration.   25,51 or DNase I combined with CAZ for another 24 h at 37 ºC to obtain 2-day biofilm. Thereafter, the biofilms on pegs were rinsed once with sterile PBS for 1 min, fixed with 99% methanol for 15 min and stained with 2% w/v crystal violet for 5 min. The excess stain was removed using running tap water and air-dried, the crystal violet stain on each peg was dissolved by immersion into 200 µL 33% (v/v) glacial acetic acid and the optical density measured at 620 nm using a microplate reader (TECAN Safire, Port Melbourne, Australia).   Confocal laser scanning microscope (CLSM) observation. To assess the impact of DNase I on CAZ efficacy in the biofilm inhibition experiment, B. pseudomallei biofilm structure and eDNA were observed on sterile 12 mm-diameter round glass coverslips held by an Amsterdam Active Attachment (AAA) model with slight modifications from a previously described method 19,51 . In brief, 1 mL of bacterial starter culture (10 8 CFU/ mL) in LB medium and 0, 0.01, 0.1 or 1 U/mL DNase I was added to each well of a 24-well plate (Costar® #3524, Corning, NY, USA). The coverslips were allowed to develop biofilm at 37 °C for 24 h. The coverslips were then washed once with sterile PBS, pH 7.4 and further incubated in fresh LB medium with DNase I, 512 µg/mL CAZ or the mixture of both agents for another 24 h. The 2-day biofilms on the coverslips were rinsed three times with sterile PBS prior to staining with 50 µg/mL fluorescein isothiocyanate-concanavalin A (FITC-Con A) (Sigma-Aldrich, Saint Louis, Missouri, USA). FITC-ConA binds α − D-mannose or α − D-glucose that are present in various sugars, glycoproteins and glycolipids including microbial cell walls (representing biofilm biomass, green) and 2 µM TOTO-3 (Thermo fisher Scientific, Oregon, USA), which binds eDNA (red) for 20 min. Separately, the viability of biofilm cells was examined using 3.34 µM/mL SYTO 9 and 5 µg/mL propidium iodide (PI) (Invitrogen, Thermo fisher Scientific, Oregon, USA) staining for 15 min. The biofilms were subsequently fixed with 2.5% glutaraldehyde in PBS for 3 h before washing with sterile PBS 3 times and air-dried for 24 h at room temperature. The biofilm structure and eDNA were visualized under a confocal laser scanning microscope (CLSM, LSM 800, Carl Zeiss, Jena, Germany). The excitation/emission maxima for these dyes were approximately 495/519 nm for FITC-ConA, 261/661 nm for TOTO-3, 483/500 nm for SYTO 9 and 305/617 nm for PI. The biofilm intensity was analyzed by z-stack processing using Zen blue software 19,53 . Biomass of adherent cells and eDNA quantity were calculated from 18 CLSM images using the COMSTAT computer program 54 . The biofilm cell viability was presented as live/dead ratio. Statistical analysis. Statistical analyses were performed using SPSS software, version 23 (SPSS Inc., Chicago, IL, USA). Data were analyzed for statistical significance using the one-way ANOVA followed by Tukey post-hoc test, or Games-Howell post-hoc test to correct for variance heterogeneity. The levels required for statistical significance were *p < 0.05 and **p < 0.001.

Data availability
The datasets used and/or analyzed during the current study available from the corresponding author on reasonable request.